Abstract
Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.
Highlights
Factor VIII (FVIII) is an essential cofactor in the blood-clotting cascade
This volume can accommodate two FVIII molecules and part of the outer lipid layer of the lipid nanotubes (LNT)’s membrane, as the volume and surface area corresponding to the FVIII-3D structure (3CDZ) low-pass filtered to 25 Å are equal to 403.1 × 103 Å3 and 31.5 × 103 Å2, respectively and the volume and surface area corresponding to the FVIII-LNT structures in Fig. 1B, low-pass filtered to 25 Å are equal to 367.7 × 103 Å3 and 31.2 × 103 Å2, respectively
We have successfully applied single particle tomography (SPT) reconstruction to resolve for the first time the structure of membrane-bound Porcine FVIII lacking the B domain (pFVIII) helically organized on LNT without enforcing helical symmetry
Summary
Factor VIII (FVIII) is an essential cofactor in the blood-clotting cascade. Its importance is illustrated by Hemophilia A, a mild to severe X-chromosome linked bleeding disorder, caused by defective or deficient FVIII1,2. Porcine FVIII lacking the B domain (pFVIII) shares 86% amino acid sequence identity with hFVIII and has similar coagulation activity, forming functional FVIIIa-FIXa complexes with human FIXa in vivo[28,29]. The pFVIII expression yield is 10 to 14 fold higher than for the human analogue[30] and its active form (pFVIIIa) is indefinitely stable at concentration greater than 0.3 mM and pH = 6.0, whereas hFVIIIa spontaneously dissociates after only a few minutes in vitro[20,23,31] These properties of pFVIII are ideal for direct structural studies by cryo-EM in a lipid environment and at close to physiological conditions. The FVIII-C2 domain membrane-binding interface[12,13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
